Regulation of exocytosis from rat peritoneal mast cells by G protein βγ-subunits

Abstract

We applied G protein-derived βγ-subunits to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca2+ and GTPγS over ~20-30 min. βγ-Subunits applied to the permeabilized cells retard this loss of sensitivity to stimulation (run-down) and it can be inferred that they interact with the regulatory mechanism for secretion. While α-subunits are without effect, βγ-subunits at concentrations > 10-8 M enhance the secretion due to Ca2+ and GTPγS. Unlike the small GTPases Rac and Cdc42, βγ-subunits cannot induce secretion in the absence of an activating guanine nucleotide, and thus further GTP-binding proteins (likely to be Rho-related GTPases) must be involved. The enhancement due to βγ-subunits is mediated largely through interaction with pleckstrin homology (PH) domains. It remains manifest in the face of maximum activation by PMA and inhibition of PKC with the pseudosubstrate inhibitory peptide. Soluble peptides mimicking PH domains inhibit the secretion due to GTPγS and block the enhancement due to βγ-subunits. Our data suggest that βγ-subunits are components of the pathway of activation of secretion due to receptor-mimetic ligands such as mastoparan and compound 48/80.